Heavy metals bioaccumulation and its impact on fatty acids composition in
the benthic crustacean mud crab (Scylla paramamosain) and associated human health risk

Introduction：

Over recent decades, accelerating industrial development and expanding human activities have emerged as major drivers of environ- mental degradation, with aquatic ecosystems among the most severely affected (A. K et al., 2023; Kabir et al., 2023; Ogidi and Akpan, 2022). Heavy metals (HMs) pose a persistent threat to aquatic ecosystems due to their bioaccumulative and non-degradable nature, making them major stressors in aquatic ecotoxicology; therefore, understanding how HMs exposure alters lipid metabolism in benthic organisms such as mud crabs is essential for assessing ecological and human health risks in estuarine environments (Edo et al., 2024; Jeong et al., 2023; Verma

et al., 2023). Heavy metals such as lead (Pb), mercury (Hg), and cad- mium (Cd) accumulate in aquatic organisms and biomagnify across food webs. This accumulation damages cell membranes, increases oxidative stress, and disrupts energy homeostasis in exposed species (Aureliano et al., 2023; Mishra et al., 2023; Yang et al., 2024; Zhou et al., 2023). These pollutants enter the human diet through contaminated aquatic species and cause serious chronic health effects, including cadmium- induced renal failure, lead-related respiratory impairment, and mercury-associated neurotoxicity and cancer (Ali and Khan, 2018; Habib et al., 2024; Khalid et al., 2018).
Crustaceans, particularly mud crabs, show high vulnerability to HMs because their sedentary lifestyle promotes the accumulation of

pollutants from contaminated sediments (Truchet et al., 2022; Waqas et al., 2024). Mud crabs are widely consumed in countries such as China, Malaysia, and Vietnam, and their contamination therefore raises serious public health concerns (Liew et al., 2023; Rahman et al., 2020; Zhang et al., 2022a). Crabs provide high nutritional value and remain popular among consumers; however, contamination poses significant health risks that cannot be overlooked (Wang et al., 2021; Yu et al., 2020a; Zhang et al., 2022a). Previous studies show that HMs contamination increases lipid accumulation, lipid peroxidation, oxidative stress, and mitochondrial dysfunction in aquatic organisms, further exacerbating their health impacts (Banaee et al., 2024; Jeong et al., 2023).
Fatty acids play a central role in energy storage, maintenance of cell membrane structure and function, and serve as important biomarkers for assessing toxicity (de Almeida Rodrigues et al., 2022; Kodali et al., 2020; Koelmel et al., 2020). Heavy metals and other pollutants disrupt fatty acid composition and lipid metabolism, impairing physiological function in exposed species such as crabs (de Almeida Rodrigues et al., 2022; Ukaogo et al., 2022). Consequently, changes in fatty acid composition serve as sensitive indicators of pollutant effects in aquatic organisms (Cha et ., baneal2020; Xiang et al., 2020). Moreover, essential fatty acids, particularly n-3 highly unsaturated fatty acids (HUFAs) such as DHA and EPA, are vital for crustacean health, sup- porting immune function, lipid metabolism, reproductive development, and cell membrane integrity (Murru et al., 2021). However, contaminant-induced oxidative stress accelerates the oxidation of n-3 HUFAs, resulting in cellular and tissue damage (Engwa et al., 2022; Yu et al., 2020b; Yu et al., 2020c). These processes disrupt normal physi- ological function in crustaceans, potentially compromising survival and propagating toxic effects through higher trophic levels, including humans. Although numerous studies have documented HMs bio- accumulation in crustaceans such as shrimp and crabs, the consequences of such contamination for fatty acid composition in wild mud crabs (Scylla paramamosain) remain poorly characterized.
In this study, we address this knowledge gap by investigating the associations between heavy metal bioaccumulation and fatty acid pro- files in the gills and muscles of mud crabs collected from three estuaries in Guangdong Province (Rongjiang, Huanggang, and Pearl River). In parallel, we evaluate potential human health risks associated with di- etary exposure to contaminated crabs.

2. Materials and methods
   2.1. Ethics statement
   The animal procedures were approved by the Shantou University Animal Care and Use Committee and conducted in strict compliance with institutional guidelines and the Guide for the Care and Use of Laboratory Animals.
   2.2. Study area
   Three representative estuarine sites were selected for this study: the Rongjiang River Estuary (116.605289◦ E, 23.358844◦ N), the Huang- gang River Estuary (116.991464◦ E, 23.578546◦ N), and the Pearl River Estuary (113.650544◦ E, 22.634456◦ N), all located in Guangdong Province, one of the most economically developed regions in China. As Wu et al. (2022) reported, the Pearl River represents a major source of HMs pollution in South China and is therefore of particular environ- mental significance within Guangdong Province. Another important estuarine region in this area is Shantou Bay, which extends through Shantou City in Guangdong Province. Shantou Bay serves as a key center for fisheries, an international port, and a rapidly expanding urban area with a dense population (Zhang et al., 2022b). The Rongjiang River serves as the primary freshwater input to Shantou Bay and acts as a major conduit for land-based pollutants, including HMs, originating from upstream urban, industrial, and agricultural activities (Shi et al.,

2016). The Huanggang River flows through Raoping County in Chaoz- hou City, eastern Guangdong, and extends approximately 87.2 km in length. It serves as a primary water source for industrial, domestic, and agricultural activities, supporting a population of about one million. However, increasing inputs of domestic sewage, agricultural runoff, and industrial effluents have substantially degraded the river's water quality. Over the past few decades, rapid industrial growth and urbanization in Guangdong have intensified heavy metal contamination of aquatic en- vironments (Shi et al., 2018), with consequent adverse effects on the health and survival of aquatic organisms.
2.3. Sample collection
A total of 120 mud crabs, with a mean body weight of 144.11 ± 11.77 g, were collected from the Rongjiang, Huanggang, and Pearl River estuaries. From each estuary, 40 crabs (20 males and 20 females) were selected and immediately transported to the Marine Biotechnology and Genetic Breeding Laboratory at Shantou University. Prior to dissection, the crabs were rinsed with distilled water and gently blotted dry with paper towels. Tissues, including gills and muscle, were then collected, placed into 20 mL plastic containers, and stored at _ 80 ◦ C until further analysis.
2.4. Samples preparation and analysis
The crabs were dissected, and gill and muscle tissues were subjected to microwave-assisted digestion using polyethylene terephthalate (PET) Teflon vessels. Briefly, 1 g of each tissue sample was placed into a Teflon vessel, and 10 mL of concentrated nitric acid (67%, sub-boiling distilled; DST-1000, Savillex) was added. Digestion was carried out using a CEM Mars 6 microwave digestion system at 180 ◦ C for 15 min, following U.S. Environmental Protection Agency Method 3052 (EPA, 2021). Following digestion, the samples were diluted 50-fold with deionized water and analyzed using inductively coupled plasma optical emission spectrom- etry (ICP-OES). Analytical accuracy was verified using the certified reference material DOLT-5, which was included in both digestion and analytical batches. Elemental recoveries for DOLT-5 ranged from 80% to 120% across all target elements, confirming the reliability of the analytical procedure. The validation parameters, such as precision, ac- curacy, and selectivity for ICP-OES were in line with established guidelines for elemental analysis (Yazman et al., 2025).
2.5. Quantification of fatty acids and their quality indices
2.5.1. Fatty acid methyl esters (FAME) preparation
Fatty acids in the muscle and gill tissues were quantified using a revised protocol based on the method of Yuan et al. (2021). Fatty acid methyl esters (FAMEs) were prepared from approximately 120 mg of thawed muscle and gills tissue using a modified derivatization protocol. Briefly, 1 mL of a methyl tricosanoate internal standard solution (1 mg mL_ 1) was added to a 12 mL Teflon-sealed tube and evaporated under a gentle nitrogen stream at 25 ◦ C. The tissue sample was then homoge- nized with 3 mL of a methanolic esterification reagent (0.25 mg mL_ 1; CH3OH:H2SO4, 99:1, v/v) containing 0.025 g L_ 1 butylated hydrox- ytoluene (BHT), vortexed for 10 min, and incubated at 80 ◦ C for 4 h. After cooling to room temperature, 1 mL of n-hexane was added and the mixture was gently agitated for 1 min. Phase separation was induced by adding 1 mL of ultrapure water, after which the organic (upper) phase was collected, filtered, and concentrated under nitrogen. The resulting FAME extract was reconstituted in 500 μL of n-hexane and stored at _ 20 ◦ C until GC–MS analysis.
2.5.2. Gas chromatography-mass spectrometry (GC–MS)
Fatty acid methyl esters (FAMEs) were analyzed using an Agilent 7890B gas chromatograph coupled to a 5977 A mass selective detector (GC–MS) equipped with a DB-WAX capillary column. Ultra-high-purity

helium was used as the carrier gas at a constant flow rate of 1.0 mL min_ 1. Samples (0.5 μL) were injected in split mode with a split ratio of 1:20, and the injector temperature was maintained at 250 ◦ C. The GC oven temperature program was as follows: an initial temperature of 100 ◦ C, increased at 10 ◦ C min_ 1 to 200 ◦ C and held for 5 min, then ramped at 10 ◦ C min_ 1 to 230 ◦ C and held for 10 min. The MS ion source and interface temperatures were set to 240 ◦ C and 230 ◦ C, respectively. Mass spectra were acquired in full-scan mode over an m/z range of 40–500. Fatty acids were identified by comparison with mass spectra from the NIST 2011 library and by relative retention times. Quantifi- cation was performed using peak area ratios relative to methyl tricosa- noate as an internal standard, and results were expressed as mg /g dry weight. The method's accuracy, precision, and selectivity were verified through the analysis of certified reference materials (CRMs), with re- coveries consistently falling within 80% to 120% for all target fatty acids. Additionally, matrix effects were evaluated, and no significant interference was observed in the fatty acid analysis. The method's robustness was confirmed through repeated analyses, ensuring high reproducibility. The results were consistent with those reported in pre- vious studies employing similar chromatographic and mass spectro- metric techniques for fatty acid analysis (Yüksel, 2020; Yazman et al., 2025).
2.5.3. Lipid quality indices
The fatty acid profile of the muscles of mud crab was calculated using four nutritional indices: AI, TI, and FLQ and OFA (Ferna’ et .,ndezal 2007). AI, TI, and FLQ and OFA were calculated by eq. 1, 2, 3 and 4, respectively.
Al and TI were calculated by eq. 1 and 2 following (Garaffo et al., 2011; Ulbricht and Southgate, 1991).
Index of atherogenicity (AI):
AI = [C12 : 0 + (4 × C14 : 0) + C16
: 0 ])/((n _ 3PUFA + n _ 6PUFA + MUFA)] (1)
Index of thrombogenicity (TI):
TI = [C14 : 0 + C16 : 0 + C18 : 0]/[(0.5 × C18 : 1)

• (0.5 × sum of other MUFA) + (0.5 × n _ 6PUFA) (2)
• (3 × n _ 3PUFA) + n _ 3PUFA/n _ 6PUFA )]
  Flesh-lipid quality (FLQ) were calculated according to (Abrami et al., 1992);
  FLQ = 100 × (EPA + DHA)/(%of total fatty acids) (3) Hypercholesterolaemic fatty acids (OFA):
  OFA = C12 : 0 + C14 : 0 + C16 : 0 (4)
  2.6. Risks to Human health risk
  2.6.1. Estimated daily intake (EDI)
  The estimated daily intake (EDI) of heavy metals was calculated by multiplying the metal concentration in crab tissue by the ingestion rate and normalizing to the average body weight of consumers. The ingestion rate (IR) was set at 0.03 kg person_ 1 day_ 1, based on the national average daily consumption of aquatic products in China (N.B.S of PRC, 2019). The average body weight of adult consumers in China was assumed to be 61.75 kg (N.H.F.P.C. of PRC, 2015).

2.6.2. Estimated weekly intake (EWI)
The EWI was calculated using Eq. 6, considering the metal concen- tration in crab tissue, the rate of ingestion per week (IRW), and the average adult body weight (Bwa). The IRW was set at 0.21 kg/person/ week, which is seven times the daily consumption rate.

2.6.3. Target hazard quotient
The Target Hazard Quotient (THQ) was calculated using the U.S. Environmental Protection Agency (USEPA, 2011) risk assessment model to estimate non-carcinogenic health risks related to heavy metal expo- sure through crab consumption. The calculation incorporated the following parameters: metal concentrations in crab tissues, an average body weight of 61.75 kg for an adult consumer, an exposure frequency corresponding to daily consumption, and an exposure duration of 70 years, representing a typical lifetime exposure (25,550 days). The esti- mated crab ingestion rate was 0.03 kg per person per day, based on national dietary consumption data for aquatic products. To assess po- tential risk, the study used oral reference doses (RfDs), which define the maximum acceptable daily intake for each metal without causing adverse health effects. The THQ was determined by comparing the estimated exposure dose of each metal with the corresponding RfD, thus quantifying the non-carcinogenic health risk from consuming crabs. The THQ values were used to assess the relative safety of crab consumption from the studied estuarine sites, with values below 1 indicating no sig- nificant health risk

2.6.4. Hazardous index (HI)
The HI is the hazard index of all selected 13 HMs to assess dietary risk from mud crabs. The HI was calculated as the sum of 13 HMs, using eq. 8.
HI = i=1 THQi (8)
2.6.5. Target cancer risk (TR)
The TR, which indicates the cancer risk from exposure, was figure out using the materials of (EPA, 2011). TR was estimated with the following eq. 9:

To calculate the cancer risk from crab consumption, numerous fac- tors were used in accordance with USEPA (2011) standards. The metal concentrations in crabs were measured, along with The CPSo value for oral carcinogenic potency and daily crab ingestion rate (FIR). The average exposure to carcinogens was assumed to last 365 days annually for 70 years. The TR for individual metals in the crab's samples was calculated using these values. The CPSo values for each metal were based on USEPA standards, including Al (1), As (1.5), Cd (0.38), Co (0.38), Cr (0.5), Cu (0.7), Fe (1), Mn (1), Ni (1.7), Pb (0.0085), Se (0.005), and Zn (0.3).

2.7. Statistical analysis
Statistical analyses were conducted using R Studio (version 3.6.2). Heavy metal concentrations are expressed as mg/kg wet weight and presented as means ± SE (n = 4 biological replicates). Each biological replicate corresponds to pooled tissue samples from five individual crabs of the same sex and estuarine site. Differences among groups were evaluated using two-way analysis of variance (ANOVA), followed by Tukey's post hoc test, with significance levels set at * P < 0.05, ** P < 0.01, *** P < 0.001. Bar plots were generated using GraphPad Prism (version 10.1.2). Cube root transformation was applied to reduce skewness and stabilize variance in heavily skewed data, particularly for variables with wide-ranging values. Autoscaling was used to standardize the data, ensuring equal contribution from each variable by adjusting for differences in units and magnitude.

3. Results
   3.1. Heavy metal concentration in muscles and Gills
   The mean concentrations of the 13 heavy metals and metalloids in male crab muscle tissues followed the order: Zn > Mn > Cu > Fe > Al > Co > Cd > As > Se > Cr > Ni > Pb > Hg. In male crab gills, concen- trations decreased in the order: Fe > Al > Cu > Mn > Zn > As > Se > Cr > Ni > Pb > Co > Cd > Hg. In female crabs, the concentration order in muscle tissues was: Zn > Fe > Cu > Al > Mn > As > Se > Cd > Cr > Ni > Pb > Co > Hg, whereas gill tissues showed the following pattern: Mn > Cu > Fe > Al > Zn > As > Se > Pb > Ni > Cr > Cd > Co > Hg.
   Among the HMs, Mn concentrations (Fig. 1h) was significantly higher in female crabs from the Huanggang River estuary than in those from the Rongjiang River estuary (F2,6 = 17.66, P < 0.003). In addition, As concentrations were significantly higher in female crabs from the Pearl River estuary than in those from the Huanggang River estuary, and in male crabs from the Pearl River estuary than in male crabs from the Rongjiang River estuary (F2,6 = 16.55, P < 0.05) (Fig. 1 b). Similarly, Ni was significantly higher in the muscles of female crabs from the Huanggang River estuary than crabs from the remaining two estuaries (Fig. 1i) (F.2, 6 = 6.59, P < 0.05). Selenium concentrations were significantly higher in the muscle tissues of female crabs from the Pearl River estuary than in those from the Huanggang and Rongjiang River estuaries (Fig. 1k) (F.2, 6 = 47.82, P < 0.001). No significant differences in HMs concentrations were observed between male and female crab muscle tissues within the same estuarine ecosystem.
   In gill tissues, Al concentrations were significantly higher in female crabs from the Huanggang River estuary than in those from the other two estuaries (F2,6 = 94.91, P < 0.0001). Al levels were also significantly higher in female crabs than in males within the Huanggang River es- tuary. In contrast, male crabs from the Pearl River estuary exhibited significantly higher Al concentrations than females from the same site (Fig. 2a). Similarly, As concentrations were significantly higher in both male and female crabs from the Pearl River and Huanggang River es- tuaries than in those from the Rongjiang River estuary (F2,6 = 546.9, P < 0.001) (Fig. 2b). Cd concentrations were significantly higher in crabs from the Pearl River estuary than in those from the other two estuaries (F2,6 = 404.8, P < 0.001) (Fig. 2 c). Cr concentrations were significantly lower in crabs from the Rongjiang River estuary than in those from the other two estuaries (F2,6 = 62.1, P < 0.0001) (Fig. 2d).
   Similarly, Co (F2, 6 = 72.21, P < 0.0001), Cu (F2, 6 = 80.23, P < 0.0001), Fe (F.2, 6 = 50.07, P < 0.001), Mn (F.2, 6 = 106.6, P < 0.0001), Ni (F.2, 6 = 126.8, P < 0.0001), Pb (F.2, 6 = 96.8, P < 0.001), Se (F.2, 6 = 766, P < 0.001), and Zn (F.2, 6 = 26.64, P < 0.001) were substantially lower in crabs from the Rongjiang River estuary compared with those from the other two estuaries (Fig. 2e-l). Notably, Fe concentrations in crabs from the Pearl River estuary and Pb concentrations in crabs from the Huanggang River estuary were significantly higher in male crabs than in female crabs (Fig. 2g and j). Hg concentrations were also significantly higher in male crabs from the Pearl River estuary than in those from the other two estuaries (two-way ANOVA; F2,6 = 10.56, P < 0.05) (Fig. 2m).
   3.2. Fatty acid composition
   Fatty acid composition in the gills of mud crabs was generally comparable between males and females and across sampling locations. However, total fatty acids (F2,18 = 3.75, P < 0.05) and omega-3 fatty acids (ω-3; F2,18 = 6.91, P < 0.05) were significantly higher in male crabs from the Huanggang River estuary than in male crabs from the Rong- jiang River estuary (Fig. 3a, d). Similarly, the sum of EPA and DHA was significantly higher in male crabs from the Huanggang River estuary than in male crabs from the other two estuaries (F2,18 = 6.04, P < 0.05) (Fig. 3f).
4. higher in female crabs than in males across all sampled estuaries. The saturated FAs (Fig. 3h) were significantly higher in male crabs from the Huanggang River estuary than in male crabs from the Pearl River es- tuary (F2,18 = 8.54, P < 0.05). In addition, monounsaturated fatty acids (MUFA) were significantly higher in female crabs from the Huanggang River estuary than in those from the Rongjiang River estuary (F2,18 = 8.54, P < 0.05) (Fig. 3i). Omega-3 fatty acids (ω-3; F2,18 = 4.72, P < 0.05) and the sum of EPA and DHA (F2,18 = 3.83, P < 0.05) were significantly higher in male crabs from the Rongjiang River estuary compared to those from the other two estuaries (Fig. 3j, l). EPA + DHA was signifi- cantly higher in female crabs' muscles from the Pearl River estuary than from the Rongjiang River estuary.
5. 3.2.1. Individual fatty acids in muscles
6. Among saturated fatty acids in muscle tissues, stearic acid (C18:0) and arachidic acid (C20:0) were consistently higher in female crabs across all selected estuaries, whereas myristic acid (C14:0) was signifi- cantly higher only in crabs from the Rongjiang River estuary (Fig. 4a, d). Stearic acid (C18:0) concentrations were significantly higher in crabs from the Rongjiang River estuary than in those from the other estuaries (F2,18 = 29.71, P < 0.001; Fig. 4c). In contrast, palmitic acid (C16:0) was significantly higher in male crabs from the Huanggang River estuary compared with those from the other estuaries (F2,18 = 11.74, P < 0.001) (Fig. 4b).
7. Monounsaturated fatty acids (MUFA), including 16:1n-7 and oleic acid, were consistently higher in the muscle tissues of female crabs than in male crabs across all selected estuaries (Fig. 4e, f). The concentration of 16:1n-7 in the muscle tissues of female crabs from the Huanggang River estuary was significantly higher than that in female crabs from the Pearl River estuary (F.2,18 = 5.137, P < 0.05). Oleic acid levels were significantly higher in crabs from the Huanggang River estuary compared to those from the Rongjiang River estuary. Additionally, oleic acid concentrations were also higher in the muscles of female crabs from the Pearl River estuary than in those from the Rongjiang River estuary (F2,18 = 18.76, P < 0.001). Eicosenoic acid (20:1n-9) was significantly higher in the muscles of female crabs compared to their male counter- parts, with the highest concentrations observed in female crabs from the Huanggang River estuary (F2,18 = 17.47, P < 0.001) (Fig. 4g).
8. Among the ω-6 fatty acids, γ-linolenic acid (GLA; 18:3n-6) was significantly higher in the muscle tissues of female crabs than in male crabs from both the Rongjiang and Huanggang River estuaries (Fig. 4i). Arachidonic acid (ARA; 20:4n-6) concentrations were significantly higher in the muscles of female crabs from the Pearl River estuary, as well as in female crabs from the Huanggang River estuary (F2,18 = 13.90, P < 0.001). In addition, eicosadienoic acid (20:2n-6) levels in crab muscle from the Rongjiang River estuary were significantly higher than those from the Huanggang River estuary (F2,18 = 15.75, P < 0.001) (Fig. 4j).
9. The levels of ω-3 fatty acids, specifically EPA and DHA, were significantly higher in the muscle tissues of female crabs compared to male crabs across all selected estuaries (Fig. 4m, o). EPA concentrations in the muscle tissues of female crabs from the Huanggang River estuary were significantly higher than those in females from the Rongjiang River estuary, whereas the opposite pattern was observed in male crabs (F2,18 = 15.06, P < 0.001). Similarly, docosapentaenoic acid (DPA; 22:5n-3) levels in crab muscle were significantly higher in individuals from the Huanggang River estuary than in those from the other two estuarine sites (F2,18 = 21.41, P < 0.001) (Fig. 4n). In contrast, α-linolenic acid (ALA; 18:3n-3) concentrations in the muscle tissues of female crabs from the Rongjiang River estuary were significantly higher than those in fe- males from the Huanggang River estuary (F2,18 = 7.36, P < 0.05) (Fig. 4l).
10. 3.2.2. Individual fatty acids in gills
11. In gill tissues, male crabs from the Huanggang River estuary exhibited significantly higher levels of saturated fatty acids (C14:0,